PEPTIDES EXHIBITING OXYTOCIN ANTAGONISTIC ACTIVITY

Abstract: A peptide having formula (I) wherein Mpa is 3-mercaptopropionic acid residue (-S-CH2-CH2-CO-); X is D-tryptophan (D-Trp) or beta -(2-Naphtyl)-D-alanine (D-Nal); Ile is isoleucine; Y is alloisoleucine (alloIle) or (S)-2-Amino-3-ethyl-pentanoic acid (Ala( beta -Et2)); Asn is asparagine; alpha -Abu is alpha -aminobutyric acid residue (II) and N alpha MeOrn is N alpha -methyl-ornithine, is disclosed. The peptides may be used as an active ingredient in a medicament, especially in a pharmaceutical composition for therapeutic treatment of excessive uterus muscle contractions.

Description: PEPTIDES EXHIBITING OXYTOCIN ANTAGONISTIC ACTIVITY The present invention relates to new peptides exhibiting oxytocin antagonistic activity. The peptides are built up of 7 amino acid residues and comprises an intramolecular ring structure. They can be used as active ingredients in pharmaceutical compositions for the inhibition of excessive uterus muscle contractions. These constitute the background for painful menstruations and premature labour. Background It has been previously shown (EP-A-0 112 809) that modifications of the vasotocin molecule in positions 1, 2, 4 and 8 give analogues, which strongly inhibit uterine contractions both in animal and human tests (Melin et al, J. Endocrinol. Ill, 125, 1986). These analogues have been shown to antagonize oxytocin or vasopressin induced contractions, and in clinical trials one analogue has been shown to counteract excessive uterus contractions in connections with painful menstruations and premature labour. (Akerlund, Acta Obst. Gynecol. Scand, 66, 459, 1987, Akerlund et al, Br. J. Obst. Gynecol, 94, 1040, 1987) . The above mentioned vasotocin analogues do not have any side effects, but they have a limited biological half-life and thus give a rather short effect duration. The enzymatic stability of the molecule, and thus the duration of the effect, is of major clinical importance at a single administration. Since the effect duration of these vasotocin analogues is comparatively short and the therapeutic dose is rather high, they have hitherto been administered intravenously in hospitals only. In order to be able to utilize oxytocin antagonists in non- institutional care, it is necessary that the properties of the molecules with regard to the effect duration and bioavailability make them suitable for non-parenteral administration such as oral and/or intranasal administration. This puts special demands on the enzymatic stability of the molecules and the ability of the molecules to penetrate biological membranes, such as nasal and gut mucous membranes. Appart from physical chemical properties one factor of importance for the penetrating ability is the size of the molecule. Thus, it is well-known that absorption through the gut mucousa increases with reduced molecular size. In WO 92/00996 is shown that derivatives of pituitary posterior lobe hormones built up of 8 amino acids has a better bioavailability after oral ingestion than the corresponding derivative having 9 amino acids. However, the single derivative therein which is built up of 7 amino acids gave potency and effect duration results that were comparable to the reference substance having 9 amino acids, but approximately three times poorer results than the corresponding derivative having 8 amino acids. The peptides according to the present invention are built up of only 7 amino acids residues. Unexpectedly they exhibit comparatively high antagonistic activity and substantially longer effect duration than any of the molecules tested in WO 92/00996. Thus, the chances are good for a high absorption to the blood after intranasal or oral administration, which has been verified for two compounds (Peptide 3 and 4) in intranasal and oral animal models. Description of the invention The present invention comprises a new peptide of the formula: Mpa-X-Ile-Y-Asn-α-Abu-NαMeOrn-NH 2 1 I wherein Mpa is 3-mercaptopropionic acid residue (-S-CH 2 -CH 2 -CO-) X is D-tryptophan (D-Trp) or β- (2-Naphtyl) -D-alanine (D-Nal) lie is isoleucine Y is alloisoleucine (allolle) or (S) -2-Amino-3-ethyl-pentanoic acid (Ala(β-Et 2 )) Asn is asparagine α-Abu is α-aminobutyric acid residue (-NH-CH-CO-) I CH 2 I CH 2 I and NαMeOrn is Nα-methyl-ornithine. One aspect of the invention is directed to a peptide according to the invention for use as an active ingredient in a medicament. Another aspect of the invention is directed to pharmaceutical compositions, which comprise at least one of the peptides according to the invention as active ingredient (s) in combination with pharmaceutically acceptable additives and/or diluents, and optionally enhancers. As pharmaceutically acceptable diluent preferably isotonic saline solution may be used. As to the other pharmaceutically acceptable additives, such can be found in the literature, e.g. the US Pharmacopoeia, and these additives shall be chosen in conformity with the specific form of the composition for a specific administration route. Enhancers which may be used are any enhancers known to facilitate absorption of a drug through a mucous membrane. Examples of absorption enhancers in nasal drug delivery has been disclosed by Merkus, F.W.H. . et al in the Journal of Controlled Release, 24 (1993) 201-208. Among the absorption enhancers mentioned are surfactants, bile acids, fusidates, fosfolipides and cyclodextrins. A composition according to the invention can be in a form which is suitable for intravenous, intranasal or oral administration. A form which is suitable for oral administration may be a tablet which is taken orally and which preferably is coated with a layer which is dissolved mainly in the intestines so that the active ingredient can be absorbed through the intestinal mucous membrane. An embodiment of this aspect of the invention is directed to a pharmaceutical composition according to the invention for use in therapeutic treatment of excessive uterus muscle contractions. Yet another aspect of the invention is directed to a method of counteracting excessive uterus muscle contractions, whereby a pharmaceutical composition according to the invention is administered in a therapeutically effectiv amount to a woman who is in need of such a treatment. The peptides according to the present invention have a specific effect on the uterus muscle and totally lack agonistic effect as well as antidiuretic effect and blood-pressure effect, resulting in high specificity, which means that possible clinical side effects are minimized. Preparation of the peptides according to the invention The peptides according to the invention can be prepared in analogy with processes well known in the peptide field. For example, the compounds according to the invention may be prepared in conventional manner by incremental coupling of amino acids to one another in liquid phase, e.g. as described by Law, H.B. & Du Vigneaud, V. in Journal of the American Chemical Society 32, (1960) 4579-4581, Zhuze, A.L. , Jost, K. , Kasafirek, E. S_ Rudinger, J. in Collection of Czechoslovak Chemical Communications 22., (1964), 2648-2662, and modified by Larsson, L.-E., B Lindeberg, G., Melin, P. / Pliska, V. in the Journal of Medicinal Chemistry 21, (1978), 352-356. The coupling of the amino acids to one another, whereby so-called peptide bonds are formed, may also be carried out by use of a solid phase (usually a resin) as starting material, to which the C-terminal of the first amino acid is coupled, whereupon the C-terminal of the next amino acid is coupled to the N-terminal of the first amino acid etc. The ring closure is performed as the last step of synthesis after or before the release of the complete peptide from the solid phase. In the following examples this so-called solid phase technique has been utilized in accordance with the disclosure according to Merrifield, R.B., J. Am. Chem. Soc. (1963), 35., 2149, Merrifield, R.B. Biochemistry (1964), 2, 1385 and Kδnig, W. , Geiger, R., Chem. Ber. (1970), 103. 788. General description of synthesis The peptides disclosed in the following examples were synthesized using the solid phase technique (J. M. Stewart, J.D. Young. Solid Phase Peptide Synthesis, Pierce Chemical Company) . The peptides were purified by liquid chromatography (reversed phase) . The stationary phase was composed of Kromasil®, 13 μ or 5 μ, 100 A, C 18 or C 8 (EKA Nobel, Sweden) and the mobile phase was acetonitrile/water having 0.1 % trifluoroacetic acid. Those fractions containing pure product (HPLC analysis) were pooled, evaporated and the product was freeze-dried from water. The purity and structure of the peptides were determined by HPLC, amino acid analysis and FAB-MS. The trifuntional amino acids were protected as follows: Fmoc-D-Trp(Boc) -OH Fmoc-Asn(Trt) -OH Fmoc-homoCys(CH 2 CH 2 C00t-Bu) -OH F oc-NαMeOrn(Pht) -OH The two last mentioned derivatives are not commercially available. Fmoc-homoCys(CH 2 CH 2 COOt-Bu) -OH was synthesized according to a publication of E. Prochazka et al, Collect. Czech. Chem. Commun. 1992, 52, 1335. Fmoc-NαMeOrn(Pht) -OH was synthesized in the same way as the corresponding lysine derivative according to a publication of R. M. Freidinger et al, J. Org. Chem. 1983, 4£, 77. (R,S) -2-amino-3-ethyl pentanoic acid was synthesized according to a publication of K. Ersler et al, Collect. Czech. Chem. Commun. __X, 1966, 4563. The Fmoc derivatives of (R,S) -2-amino-3-ethyl pentanoic acid and alloisoleucine were synthesized according to a publication of P. B. W. Ten Kortenaar, et al, Int. J. Peptide Protein Res., 1986, 21, 398. The following abbreviations have been used: TBTU = 2- (1-H-benzotriazol-l-yl) -1,1,3,3-tetramethyl-uronium- tetrafluoroborate MBHA = 4-methyl-benzhydrylamine Boc = t-buthyloxycarbonyl Fmoc = 9-fluorenylmethoxycarbonyl NMP = N-methylpyrrolidone TFA = Trifluoroacetic acid DIC = Diisopropyl carbodiimide HOBt = 1-hydroxybenzotriazole DBU = l , 8 -diazabicyclo [5 .4 . 0] undec-7-ene DMF = Dimethylformamide DIPEA = Diisopropylethylamine Pht = Phthaloyl Trt = Triphenylmethyl (Trityl) E am le 1 Peptide 3 ( -NαMeOrnNH 2 ) CH 2 -CH 2 -CH 2 -NH 2 CH 2 CH 2 CO-D-Trp- Ile-alloIle-Asn-NH-CH-CO-N-CH-CO-NH 2 CH — CH 2 CH 3 The peptide was synthesized according to a mixed Boc/Fmoc methodology on solid phase. Activation of the amino acids was made with TBTU/HOBt. The resin was of MBHA-type with the loading of 0.65 mmole/g, and 0.33 g was used for each synthesis. The first amino acid was NαBoc protected. The Boc group was removed with 50 % trifluoroacetic acid in dichloromethane. The rest of the synthesis followed the Fmoc strategy, i.e. Nα-Fmoc protected amino acids were coupled to the free amino group of the previously coupled amino acid. The Nα-Fmoc group was removed with 20 % piperidine in NMP. When all the amino acids had been coupled to the resin, the resin was treated with 20 % piperidine in NMP, followed by 50 % TFA in dichloromethane. The peptide chain was thereafter cyclized while it was still attached to the resin, from D-Trp 2 to homoCys(CH 2 CH 2 COOH) 6 by activating with TBTU/HOBt, i.e. between positions 1 and 2. The peptide was cleaved from the resin with liquid hydrogen fluoride-thiocresol- cresol-dimethylsulfide in the ratio of 8-0.25-0.75-1 at 0°C. After evaporation of the hydrogen fluoride, the resin was suspended in diethyl eter, filtrated and washed with additional diethyl eter. The precipitated peptide was recovered from the resin by dissolving in acetic acid. The acetic acid was evaporated and the residue freez-dried from water. The freez- dried product was dissolved in ethanol, whereupon hydrazine hydrate was added in a molar excess. The solution was stirred over night at room temperature, whereupon it was acidified with H 3 0 + and evaporated, and freez-dried from water. The product was purified in the same way as above. Yield: 4 mg. Purity (HPLC) = 95 %. Example 2 Peptide 4 CH CH CO-D-Nal-Ile-alloIle-Asn-NHCHCO-NαMeOrn-NH, ■ CH 2 —CH The peptide was synthesized according to Fmoc methodology on solid phase. Activation of the amino acids was made with DIC/HOBt. The resin was of TentaGel-S-type with RAM-linker (Rapp Polymere S 30023) . The Nα-Fmoc-groups were removed by 2% DBU in DMF. The peptide was cleaved from the resin and deprotected with TFA/ethanedithiol/anisole in the ratio of 95:2.5:2.5. The reaction mixture was filtered and the filtrate was concentrated. The peptide was precipitated with diethyl ether. Then the peptide was cyclized between the positions 1 and 2 by activation with TBTU/HOBt/DIPEA in DMF. After the cyclization NαMeOrn(Pht) was deprotected by the addition of hydrazine hydrate. Following stirring overnight at room temperature, the solution was neutralized and evaporated. The product was purified as disclosed above. Yield: 11 mg Purity (HPLC) : 99% Example 3 Peptide 5 CH 2 CH 2 CO-D-Nal-Ile-Ala(β-Et 2 ) -Asn-NHCHCO-NαMeOrn-NH 2 1 2 3 4 5 6 7 S————————————————————CH —CH The peptide was synthesized according to the same methodology as in Example 2. At the purification performed as above the analogues with the R and S isomers of Ala(β-Et 2 ), respectively, were separated. The S isomer (Peptide 5) was eluated after the R isomer. The enantiomeric forms of the respective peptide analogue was determined by first hydrolyzing and then derivatizing the amino acids, and analyzing the mixture on a chiral column by a gas chro athographic method. Yield: 10 mg Purity (HPLC) : 98% PHARMACOLOGICAL TESTS In vivo tests, rat Sprague Dawley rats (250 g) in natural estrous were anaesthesized with Inactin (0.5 mg/lOO g body weight i.p.) . The activity of the myometrium was measured with the aid of a catheter which had been fixed in the uterus cavity and which had been filled with modified Locke's solution. The catheter was connected to a Statham P23d force transducer and the contractions were registered on a Grass polygraph (model 7D) . Antagonist tests. Inhibitory Dose (I.P.) [I.D. = that antagonist dose which inhibits an agonist dose (2 x) to an effect corresponding to the effect of half the agonist dose (x) ] . At first, the dose-effect curve for oxytocin (2.10 "4 - 5.10 "3 μmole/kg) was carried out. Such an oxytocin dose (2 x) is selected which gives an effect corresponding to an intraluminar contraction pressure of 10-30 mg Hg and which lies on the linear part of the dose-effect curve. The effects are measured as the net values of the integrated curve measured during 15 minutes after the injection. Then, the effect (eff x) of the agonist is calculated for its half dose (x) . Thereafter at least two doses of antagonist (Peptide 1-4) are injected in combination with the agonist dose (2 x) . By interpolating the dose-effect curve for the inhibition, the antagonist dose corresponding to the effect (eff x) of the agonist dose (x) , i.e. the I.D. dose, is obtained. The results are shown in Table I. Antagonist tests, effect duration. (Dura ion, mϊn Q.75) Such a dose of the agonist is selected (5.10 "4 - 5.10 "3 μmole/kg) which gives an effect (the effect is measured during a 15 minutes period after agonist and antagonist administration, respectively, the contraction curve being integrated) corresponding to approximately 50 % of the maximum effect (ED 50 ) . Then a dose of the antagonist (Peptide 1-3) 0.8 - 4.10 "8 mole/kg, which in combination with the agonist dose gives at least 50 % inhibition of the effect of the agonist dose only, is administered. Then, only the agonist dose is injected at 20 minutes intervals, the inhibition effect successively declining. By interpolating, the period of time from administration of the inhibitor to when 75 % of the inhibition of the agonist effect has ceased, is obtained. The results are shown in Table I. IΔBLEJ: Pharmacological data for oxytocin antagonists In vivo, rat Test substance ID nmol/kg Duration, min 0.75 Peptide 1 (ref.) 6.3 ± 0.8 29 ± 5 Peptide 2 (ref.) 2.2 ± 0.3 140 ± 19 Peptide 3 (inv.) 2.9 ± 0.2 248 ± 29 (preliminary 3.6 ± 0.6) Peptide 4 (inv.) 1.8 ± 0.04 257 ± 20 Peptide 5 (inv.) 2.7 ± 0.1 226 ± 41 Peptide 1 (ref.) = Peptide according to EP 0 112 809 having the formula Mpa-D-Tyr(Et) -Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH 2 1 I Peptide 2 (ref.) = The tested peptide which according to WO 92/00996 has the longest duration, having the formula Mpa-D-Phe(p-Et) -Ile-Thr-Asn-α-Abu-Pro-Orn-NH 2 Peptide 3 (inv.) = The peptide according to the invention having the formula Mpa-D-Trp-Ile-alloIle-Asn-α-Abu-NαMeOrn-NH 2 J I Peptide 4 (inv.) = The peptide according to the invention having the formula Mpa-D-Nal-Ile-alloIle-Asn-α-Abu-NαMeOrn-NH 2 Peptide 5 (inv.) = The peptide according to the invention having the formula Mpa-D-Nal-Ile-Ala(β-Et 2 ) -Asn-α-Abu-NαMeOrn-NH 2 J 1 It is evident from Table I that the Peptides 3, 4 and 5 according to the invention have a substantially prolonged effect duration with retained or improved (lower ID values) potency compared to the reference substances Peptide 1 and Peptide 2, respectively.

Claims: CLAIMS 1. Peptide having the formula Mpa-X-Ile-Y-Asn-α-Abu-NαMeOrn-NH 2 i I wherein Mpa is 3-mercaptopropionic acid residue (-S-CH 2 -CH 2 -CO-) X is D-tryptophan (D-Trp) or β- (2-Naphtyl) -D-alanine (D-Nal) lie is isoleucine Y is alloisoleucine (allolle) or (S) -2-Amino-3-ethyl-pentanoic acid (Ala(β-Et 2 )) Asn is asparagine α-Abu is α-aminobutyric acid residue (-NH-CH-CO-) NαMeOrn is Nα-methyl-ornithine. 2. Peptide according to claim 1 for use as an active ingredient in a medicament. 3. Pharmaceutical composition, c h a r a c t e r i z e d in that it comprises at least one peptide according to claim 1 as active ingredient(s) , together with pharmaceutically acceptable additives and/or diluents, and optionally enhancers. 4. Pharmaceutical composition according to claim 3, which is in a suitable administration form for intranasal administration. 5. Pharmaceutical composition according to claim 3, which is in a suitable administration form for intravenous administration. 6. Pharmaceutical composition according to claim 3, which is in a suitable administration form for oral administration. 7. Pharmaceutical composition according to any one of claims 3-6 for use in therapeutic treatment of excessive uterus muscle contractions. 8. A method of counteracting excessive uterus muscle contractions, in which a pharmaceutical composition according to any one of claims 3-6 is administered in a therapeutically effective amount to a woman in need of such treatment.

Inventors: AURELL CARL-JOHAN, MELIN PER, NILSSON ANDERS, TROJNAR JERZY, AURELL, CARL-JOHAN, MELIN, PER, NILSSON, ANDERS, TROJNAR, JERZY
Applicants: AURELL CARL JOHAN, FERRING BV, MELIN PER, NILSSON ANDERS, TROJNAR JERZY, AURELL, CARL-JOHAN, FERRING B.V., MELIN, PER, NILSSON, ANDERS, TROJNAR, JERZY
Application year: 1994
Main classification: C07K7
IPC codes: A61K38, A61P13, A61P15, C07K7